Development of a transcription-reverse transcription concerted reaction method for specific detection of human enterovirus 71 from clinical specimens.

Abstract

A transcription-reverse transcription (RT) concerted reaction (TRCR) method was developed for rapid and specific detection of EV71 from clinical specimens. This method was validated with EV71 strains from all of the known genotypes (genotypes A, B1 to B5, and C1 to C5), with detection limits of 10 to 10(3) copies, and was useful for identification of EV71 from throat swabs of patients with hand, foot, and mouth disease (HFMD).

A TRCR method for the detection of EV71 from clinical specimens. (A) Procedure and a primer-probe set for the TRCR method. Viral RNA and TRCR reagents and enzymes were preincubated at 43°C for 5 min and 2 min, respectively, and then were mixed. EV71-targeting regions and T7 promoter regions of the forward primers are colored red and blue, respectively. The positions of oxazole yellow dye in INAF probes are indicated by green asterisks. (B) Histogram of RNA samples extracted from throat swabs of HFMD or herpangina patients in Japan (total 58 samples) grouped by the numbers of copies of viral RNA.

Comparison of the nucleotide sequences of the EV71 and CVA16 genomes examined for the primers and scissor probes. Genomic sequences of the EV71 and CVA16 strains are shown with their genotypes. Nucleotide differences in EV71 genomic sequences and in primers or probes are highlighted in red (genotype A), blue (genotype B), and green (genotype C).

Comparison of the nucleotide sequences of the EV71 and CVA16 genomes for the INAF probes. Genomic sequences of EV71 and CVA16 strains are shown with their genotypes and names. Nucleotide differences in the EV71 genomic sequences and in INAF probes are highlighted in red (genotype A), blue (genotype B), and green (genotype C). Flanking nucleotides of oxazole yellow dye are colored in cyan in the INAF probe sequences. INAF probes (1, 2, and 3) and the target EV71 strains are colored in cyan, yellow, and pink, respectively. For detection of genotype C3 of EV71, INAF probes 2 and 3 were required. The amino acid sequences of EV71 and CVA16 in the INAF probe-binding region are shown below.